Part of the pathology of atherosclerosis and post-angioplasty restenosis involves the regression of vascular smooth muscle cells (VSMCs) to a less differentiated cell that is capable of proliferation and migration. To understand these processes at a molecular level, we have isolated and characterized genes encoding transcription factors that are differentially expressed in quiescent and proliferating VSMCs. Previous work in my laboratory has led to the isolation of a homeobox transcription factor gene dubbed gax from an adult aorta VSM cDNA library. In VSMCs, gax expression is rapidly repressed by growth factor stimulation, and more slowly upregulated under conditions that promote quiescence. Similarly, expression of gax is rapidly downregulated in rat carotid arteries following an injury that stimulates VSMC proliferation. Adenovirus-mediated gax overexpression markedly inhibits injury-induced intimal hyperplasia in both rat carotid artery and rabbit iliac artery models of balloon denudation. Constitutive gax expression in vitro blocks both mitogen-stimulated cell cycle activity and mitogen-directed VSMC migration. Under conditions of prolonged mitogen activation, forced gax expression ultimately leads to p53-independent apoptotic cell death. The goal of the proposed research is to use Gax as a molecular reagent to characterize mechanisms that coordinate VSMC differentiation, proliferation and death. Therefore, renewal of funding is requested to: 1) perform a mutational analysis of the Gax transcription factor to determine structure-function relationships; 2) isolate and characterize Gax-binding protein; 3) identify downstream genes regulated by Gax and determine mechanisms of transcriptional regulation; and 4) identify promoter/enhancer regulatory elements in the gax gene that confer tissue-specific expression during embryogenesis and mediate the downregulation of expression that occurs upon vascular injury.